Objective-coupled selective plane illumination microscopy

ABSTRACT

A microscope assembly ( 102 ) includes an illumination source ( 104 ) coupled to an optical assembly by a coupler ( 132 ). The optical assembly includes an objective with optics that move along an optic axis. The illumination source ( 104 ) generates a light blade ( 106 ) that illuminates a portion of a sample ( 136 ) at an illumination plane ( 110 ). The light blade ( 106 ) induces a fluorescent emission from the sample ( 136 ) that is projected through the objective optics to a detector ( 126 ). The focal plane ( 122 ) of the objective optics is fixed with respect to the illumination source ( 104 ) by the coupler ( 132 ) so that the illumination plane ( 110 ) is coincident with the focal plane ( 122 ) as the objective optics move along the optic axis ( 124 ). The objective and illumination may be rapidly scanned along the optic axis to provide rapid three-dimensional imaging while the objective and illumination may also be rapidly scanned along the optic axis ( 124 ) to provide rapid three-dimensional imaging.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made, at least in part, with funding from theNational Institutes of Health, NIH Grant 1 R01 DC005964-01A1 (Holy).Accordingly, the United States government may have certain rights inthis invention.

BACKGROUND

Many biological or medical phenomena are studied using optical imaging.Optical imaging, most often in the form of microscopy, can yieldinsights at cellular and sub-cellular levels as well as over coarserscales.

Particular proteins or other cellular components can be tagged with afluorescent dye for study by fluorescence microscopy. Alternatively,dyes that change their fluorescence depending on calcium levels, pH,membrane voltage, or other physical or chemical characteristics can beused to report on biological phenomena such as the activity of neurons.

One frequent approach to studying these phenomena is to use wide-fieldillumination, often called epifluorescence. However, this optical methodis not suited for studying individual cells in densely-labeled,three-dimensional tissues: these optical methods do not rejectout-of-focus light, resulting in a hazy and unfocused fluorescence. Thelight used in these techniques is not restricted to the region ofinterest.

Other techniques, such as confocal or two-photon microscopy, rejectout-of-focus light and achieve a higher signal-to-noise level. However,these techniques encounter serious difficulties in studying the activityof entire populations of neurons or neural circuits, largely because oftheir intrinsically slow rate of data collection and/or rapid photobleaching of the sample.

An alternative to confocal and two-photon microscopy are systems thatemploy planar illumination. These systems often are used to imagesamples that are placed in a gel or other liquid substance. The samplesare placed in a sample chamber, and a light source generates a lightplane sideways into the sample or sample chamber, such as along ahorizontal axis. A camera may be placed along a vertical axis so that itis directly above the sample chamber. The sample chamber is then rotatedor otherwise moved to image sections of the sample.

This method results in several problems. First, only small samples areeffectively sectioned, because the light might otherwise have topropagate through many centimeters of tissue. Also, because the opticsand camera are placed above the sample and the sample must be moved toobtain sectioned images, a three-dimensional image can only be acquiredslowly. Additionally, samples in the gel or other liquid move as thesample chamber moves. This can blur sample images and does not provideaccurate sample images.

Consequently, new systems and methods are needed for optically studyingand/or recording entire neural circuits in mammals. In addition, newsystems and methods are needed for optically imaging tissues for avariety of technologies, including surgical applications and otherreal-time three-dimensional microscopy.

SUMMARY

In one embodiment, a microscope assembly may include an illuminationsource coupled to an optical assembly by a coupler. The optical assemblymay include an objective with optics that move along an optic axis. Theillumination source may generate a light blade that illuminates aportion of a sample at an illumination plane. The light blade can inducea fluorescent emission from the sample that is projected through theobjective optics to a detector. In other embodiments, the light from alaser or other source propagates through free space to the optics thatcreate the light blade. The focal plane of the objective optics may befixed with respect to the illumination source by the coupler so that theillumination plane is coincident with the focal plane as the objectiveoptics move along the optic axis. In one embodiment, the coupler is amechanical arrangement that physically connects the illumination opticsto the objective, while in another embodiment the coupling is achievedby a control system that electronically insures the illumination opticsmaintain a fixed relationship relative to the focal plane of theobjective. In one aspect, the objective and illumination may be rapidlyscanned along the optic axis to provide rapid three-dimensional imaging.The microscope assembly may be tilted in some embodiments so that theillumination plane illuminates the sample at an angle, thereby improvingthe projection from the sample that is detected by the detector. In anembodiment, the illumination source may include fiber and a fiber opticassembly coupled to the objective. In this embodiment, the fiber carrieslight from a laser to the fiber optic assembly which focuses the lightto a thin light blade at the focal plane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified block diagram of a microscope system;

FIG. 2 is a simplified block diagram of another microscope system;

FIG. 3 is a simplified block diagram of yet another embodimentmicroscope system; and

FIG. 4 is a simplified block diagram of an alternate embodiment of themicroscope system.

DETAILED DESCRIPTION

Objective-coupled planar illumination restricts the illumination of asample to the focal plane of a microscope objective lens.Objective-coupled planar illumination therefore performs opticalsectioning using a thin “blade” of light to restrict illumination nearthe focal plane. The light blade generally is produced using acylindrical lens to focus light from a laser or other illuminationsource. In one implementation, an optical fiber carries the light fromthe laser to the cylindrical lens. Producing the light blade with acylindrical lens results in weak convergence in one, but not the other,dimension of the collimated output of a fiber-coupled laser.

Because of diffraction, the light blade is not arbitrarily thin. If asample has a constant thickness d, then the minimum width of the bladeis of the order √{square root over (λd)}, where λ is the wavelength oflight. In one example, a light blade's width is in the range of 5-10micrometers (μm), which will produce effective optical sectioning.

Objective-coupled planar illumination excites only the in-focus portionof the sample, and therefore has a major advantage over confocalmicroscopy in reducing phototoxicity of the sample since fewer photonsimpact the sample. Accordingly, samples may be imaged for much longerdurations without damage.

Because the entire focal plane is illuminated simultaneously, an entireimage can be acquired using objective-coupled planar illumination ratherthan a single pixel. This permits an increase in multiple orders ofmagnitude the rate at which individual images can be acquired. Unlikeconfocal or two-photon microscopy, there is no forced tradeoff betweenimaging speed, size of the imaged area, and signal-to-noise ratio. Thus,in practice, higher signal-to-noise images are obtained using this typeof planar illumination than using confocal microscopy and other methods.

FIG. 1 depicts an embodiment of a microscope assembly 102. Themicroscope assembly 102 may be used for optical sectioning, includingproducing two-dimensional images of a sample section and using theimages to reconstruct a three-dimensional image of the sample. Themicroscope assembly 102 may include an illumination source 104 thatgenerates a blade of light 106 along an illumination path 108 to anillumination plane 110. The illumination path 108 generally correspondsto an illumination axis 112 of the illumination source 104.

In one embodiment, the illumination source 104 may include fiber thatcarries a light beam to an optic assembly 114. In this embodiment, alaser (not shown) may generate the light beam. In another embodiment,the illumination source 104 may be a laser that generates a collimatedlight beam through the optic assembly 114. In still another embodiment,the illumination source 104 may include a laser that propagates lightthrough space to the optic assembly 114. In yet another embodiment, theillumination source 104 may include the optic assembly 114 that receiveslight propagated through space or another carrier, such as lightgenerated from a laser and propagated through space to the opticassembly. Other illumination sources, such as light-emitting diodes, mayalso be used.

As shown, the optic assembly 114 may include a cylindrical lens or otherlens or focusing device that focuses the light beam into the thin lightblade 106 and to the illumination plane 110. Other optic assembliesemploying other lens types, such as graded-index (GRIN) lenses, may alsobe used to focus the light to a light blade and to an illuminationplane. The microscope assembly 102 may further include an opticalassembly 114 with an optical tube 118 and an objective 120 having anobjective lens, other optics, or another focusing device capable offocusing at a focal plane 122 along an optic axis 124.

The optical assembly 114 may include a detector 126 that detects imagesprojected from the focal plane 122 along a detection path 128 throughthe objective 120. In one embodiment, the detection path 128 correspondswith the optic axis 124. An optional excitation cutoff filter 130 may beprovided that enables selective wavelengths of light to be detected bythe detector 126. Other optional filters such as dichroic mirrors may beincluded in the optical assembly 124 or the illumination source 104.

In one embodiment, the detector 126 may be a digital camera. Forexample, the detector 126 may be a charge-coupled device (CCD) camera.In another embodiment, the detector 126 may be a person with eyes suchthat the person may view an image or the sample through the microscopeassembly 102, such as through eyepieces. The detector 126 may beconnected or removably connected to the optical tube 118 in someembodiments. In other embodiments, a connection may carry projectedimages to the detector 126. In yet other embodiments, components of thedetector 118 may be distributed.

A coupler 132 may couple the illumination source 104 and the objective120 so the illumination source is fixed relative to the objective. Inone embodiment, the coupling results in an angle alpha (α) between theillumination axis 112 and the optic axis 124 that is approximatelyorthogonal. However, the coupling angle α may vary in other embodiments.The coupler 132 fixes the separation between the focal plane 122 of theobjective 120 and the illumination plane 110 of the illumination source104. The illumination plane 110 illuminates only the focal plane 122 andexcites only the in-focus portion of a sample. The thin light blade 106is, therefore, coincident with or restricted to the focal plane 122.

The coupler 132 couples the illumination source 104 to the objective 120so that as the objective is focused in or out, the illumination plane110 concurrently moves in and out with the focal plane 122 of theobjective. Moving the objective 120 along the optic axis 124 moves thecoupler 132 and the illumination source 104 along the same axis. In oneembodiment, the coupler 132 may movably couple the illumination source104 to the objective 120 of the optical assembly 114 so that theillumination source and the objective are movably fixed with respect toeach other. The microscope assembly 102 may be manufactured so that theillumination source 104 and the objective 120 are physically coupled bythe coupler 132 at a fixed coupling angle or a selectable couplingangle.

In another embodiment, the coupler 132 may couple the illuminationsource 104 and another portion of the optical assembly 114 so that theillumination source is fixed relative to the optical assembly. Thecoupling results in an angle alpha (α) between the illumination axis 112and the optic axis 124 that is approximately orthogonal in one example.However, the coupling angle α may vary in other examples.

In this embodiment, the coupler 132 may fix the separation between thefocal plane 122 of the objective 120 and the illumination plane 110 ofthe illumination source 104. The illumination plane 110 illuminates onlythe focal plane 122 and excites only the in-focus portion of a sample.The thin light blade 106 is, therefore, coincident with or restricted tothe focal plane 122.

Because of the coupling, the illumination source 104 concurrently moveswith the optical assembly 114 if the optical assembly is moved orvice-versa. In one example, the coupler 132 may physically couple theillumination source 104 to the optical tube 118. In another example, thecoupler 132 may movably couple the illumination source 104 to theoptical assembly 114 so that the illumination source 104 and the opticalassembly 114 are movably fixed with respect to each other. Themicroscope assembly 102 may be manufactured so that the illuminationsource 104 and the optical assembly 114 are coupled by the coupler 132at a fixed coupling angle or a selectable coupling angle.

In the embodiment shown in FIG. 1, the coupling 132 may be a rigidmechanical device that provides a fixed relationship between theillumination source 104 and the objective 120. In the alternativeembodiment shown in FIG. 4, both the illumination source 104 and theobjective 120 can be moved independently using an electronic controlsystem 201 to insure that the increments of the illumination source 104and the objective 120 are synchronized to maintain a fixed relationship.In particular, the electronic control system 201 may include a pair ofactuators 250 and 252 operatively associated with the objective 120 andillumination source 104, respectively. Each actuator 250 and 252 arecontrolled by a respective control signal, such as a control voltage,generated by a processor 140 which is in operative association withactuators 250 and 252. In one embodiment, the actuators 250 and 252 arepiezoelectric devices that maintain the illumination plan 110 in thefocal plane 122 of the objective 120. In other embodiments, the controlsystem 201 may include measurements of the real position of eachactuator 250, 252 to facilitate the synchronization of the controlsystems.

Because the illumination source 104 is coupled to the objective 120 oranother portion of the optical assembly 114, the entire microscopeassembly 102 (not including the sample 136 and the sample chamber 138)may be raised, lowered, and rotated without moving the sample 136 or thesample chamber 138. Only the focal plane 122 and the illumination plane110 need be moved. This enables a sample 136 to be more rapidly imagedthan prior systems in which the sample 136 itself was raised, lowered,or rotated. A higher speed three-dimensional imaging of a sample 136 maybe performed with the microscope assembly 102.

In one embodiment, the microscope assembly 102 may be tilted at a tiltangle theta (θ). In one example, the tilt angle is an angle between theoptic axis 124 and a reference plane 134. The reference plane 134 may bea reference plane 134 of a sample 136 in a sample chamber 138, ahorizontal plane, or another reference plane. In one example, the tiltangle is 45 degrees. The sample reference plane in this example isparallel to a horizontal plane. However, the tilt angle may be greateror less than 45°. In another embodiment, the tilt angle is between 30and 60 degrees, while in another embodiment, the tilt angle is between 0and 900. The microscope assembly 102 may be manufactured so that thetilt angle θ is permanently fixed or selectable.

The tilt angle and the coupling angle result in the illumination source104 emitting the light blade 106 to the sample 136 at an illuminationangle phi (φ). This angle of illumination φ results in the illuminationplane 110 being restricted to the focal plane 122. This in turn resultsin a better sample projection along the detection path 128 to thedetector 126 than if the sample was illuminated from the side, such asalong a horizontal. The detector 126 therefore obtains better resolutionof the sectioned images. In one embodiment, the illumination angle maybe approximately between 30 and 60 degrees. In another example, theillumination angle may be 45 degrees. In other embodiments, theillumination source 104 may be configured to generate the thin lightblade 106 to the sample 136 at an illumination angle when the tilt angleis zero or another angle and/or when the coupling angle is other thanapproximately orthogonal.

The microscope assembly 102 may be configured to scan in one or moredirections. In one embodiment, the microscope assembly 102 scans in ascan direction 140 along the optic axis 124. In another embodiment, themicroscope assembly 102 scans in a scan direction 142 along thereference plane 134 or another plane, such as the horizontal plane.

Scanning speed is significantly increased when scanning along the opticaxis 124 because the illumination source 104, the objective 120, and thecoupler 132 are relatively small in mass and move together along theoptic axis 124. An alternative scanning direction, for exampleapproximately along the sample reference plane 134, would also be fastif illumination source 104, the objective 120, and the coupler 132 movetogether in the scanning direction.

In one example, because the optics of the optic assembly 114 arepositioned above the sample 136 larger samples 136 may be imaged.Further, tilting the microscope assembly 102, and thereby tilting theoptical assembly 114, enables larger samples 136 to be imaged.

In one example of operation, the sample 136 may be located in a gel orliquid solution in the sample chamber 138. The coupler 132 physicallycouples the illumination source 104 to the objective 120 so that theillumination axis 112 is at an angle to the optic axis 124. In thisexample, the coupling angle α is approximately orthogonal. A tilt angleθ also may be selected. In this example, the tilt angle is approximately45°.

The illumination source 104 generates a thin light blade 106 along anillumination path 108 to an illumination plane 110 that is fixed on thesample 136 at a focal plane of the objective 120. The illumination plane110 induces fluorescence in the sample 136 at the focal plane 122. Theemitted light is detected through the objective 120 along the detectionpath 128 at the detector 126. The sample 136 is repeatedly imaged inthis fashion in a scan direction 140 along the optic axis 124 until eachsection of the sample 136 is optically imaged. In another example ofoperation, the sample 136 is repeatedly imaged in another scan direction142 until each section of the sample 136 is optically imaged.

FIG. 2 depicts another embodiment of a microscope assembly, designated102A. The microscope assembly 102A may include optional components forsome embodiments, such as barrier filters (130).

The optical assembly 114 may include an objective positioner 202 thatmoves the objective 120 in and out along the optic axis 124. In someembodiments, the objective positioner 202 moves the coupled objective120 and illumination source 104 in the scan direction 140. The objectivepositioner 202 may be a manual positioner. In other embodiments, theobjective positioner 202 may be controlled by a processor 204 or anotherprocessing device.

A rotational positioner 206 rotates the microscope assembly 102A about arotational axis 208, such as the optic axis 124, the illumination axis112, or another axis. The rotational positioner 206 rotates themicroscope assembly 102A in one or more selected directions, such asclockwise and counterclockwise. In some embodiments, the rotationalpositioner 206 may be a manual positioner, while in other embodiment therotational positioner 206 may be controlled by a processor 204 oranother device. As further shown, a vertical positioner 210 raises andlowers the microscope assembly 102A. In some embodiments, the verticalpositioner 210 may be a manual positioner, while in other embodimentsthe vertical positioner 210 is controlled by a processor 204 or anotherdevice.

In one embodiment, a scan direction positioner 212 may move themicroscope assembly 102A in the alternate scan direction 142, such as inthe horizontal or in another scanning direction. In other embodiments,the scan direction positioner 212 may be a manual positioner, while inother embodiments the scan direction positioner 212 may be controlled bya processor 204 or another device. The alternate scan directionpositioner 212 is an optional component.

The processor 204 receives two-dimensional images from the detector 126.In addition, the processor 204 processes the two-dimensional images tocreate a three-dimensional image of the sample 136. In some embodiments,the processor 204 may control the detector 128 and its operation,including the detection and collection of images. In one embodiment, theprocessor 204 includes volatile and non-volatile memory that storesoperating software or firmware, imaging software or firmware, andimages. The processor 204 is an optional component.

The processor 204 may control how and when the objective positioner 202changes the position of the microscope assembly 102A. In otherembodiments, the processor 204 may not control the position of theobjective positioner 202.

Referring back to FIG. 2, the microscope assembly 102A may be coupled toa frame 214 that enables the rotational positioner 206 and the verticalpositioner 210 to change the position and/or orientation of themicroscope assembly 102A relative to the reference plane 134 of thesample 136 or another orientation reference. The frame 214 may becoupled to the optical assembly 114 or another portion of the microscopeassembly 102A. For example, the frame 214 may be fixedly, movably, orremovably coupled to illumination source 104, the optical tube 118, thedetector 126, the coupler 132, and/or another portion of the microscopeassembly 102. Other examples exist. The frame may be configured to reston or be attached to a table, workbench, other structure, or surface.

The frame 214 is optional in some embodiments. In some embodiments, theframe does not exist, and the rotational positioner 206 and verticalpositioner 210 may operate on another portion of the microscope assembly102 to change the orientation of the microscope assembly relative 102 tothe reference plane 134 of the sample 136 or another orientationreference.

FIG. 3 depicts another embodiment of a microscope assembly 102B.

FIG. 3 having a processor 204A that controls the rotational positioner206A and the vertical positioner 210A. The processor 204 receivesorientation instructions for the microscope assembly 102B and controlshow and when the rotational positioner 206 and/or the verticalpositioner 210 changes the position and/or orientation of the microscopeassembly 102B. The orientation instructions may be received from one ormore data entry devices, another processor, one or more sensors, one ormore switches, or other devices. A frame 214 is not depicted in theembodiment of FIG. 3. However, the embodiment of FIG. 3 may include theframe 212 of FIG. 2 in one example. In this example, the rotationalpositioner 206 and the vertical positioner 210 operate to change theposition and/or orientation of the microscope assembly 102 on or withreference to the frame 214.

It will be appreciated that the microscope assembly 102 has manybenefits. Because the illumination source 104 is coupled to theobjective, as the objective is focused in or out, the plane ofillumination moves with the focal plane. This ensures that theillumination plane always stays in the focal plane.

The microscope assembly 102 avoids the problems with blur andout-of-focus backgrounds in images that make the epifluorescence imagehard to interpret. High signal-to-noise images are obtained by usingthis system. Little photo bleaching occurs when recording images by themicroscope assembly 102.

Since the entire focal plane is illuminated simultaneously, images canbe acquired rapidly, with theoretical limits in the range of a millionframes per second. The limitation on the number of frames imaged is onlyrestricted by current camera technology.

Because the illumination source 104 is coupled to the objective 120, theentire microscope assembly may be raised, lowered, or rotated so thatsamples may be rapidly scanned in three dimensions without having tomove the sample or the sample chamber. Since the illumination source andthe objective are positioned with one or more positioners, samples maybe more rapidly scanned. Positioning the microscope assembly up, down,and rotationally with respect to the sample also enables large samplesto be imaged.

Moving the microscope assembly 102 rather than the sample results in thesample being more stable in the sample chamber, which results in agreater resolution of a sectional image. High-speed optical sectioningis thereby improved for the three-dimensional image reconstruction.

The systems and methods may be used for optical imaging in a variety oftechnologies, including surgical applications and other real-timethree-dimensional microscopy. The systems and methods may also be usedfor imaging thick tissues and other tissues and for non-tissue imaginguses.

Those skilled in the art will appreciate that variations from thespecific embodiments disclosed above.

1. A system for imaging comprising: an illumination source (104)configured to generate a thin light blade (106) to an illumination plane(110) and comprising fiber configured to carry a beam of light and anoptic assembly (114) configured to focus the beam of light into the thinlight blade (106) and to the illumination plane (110); an objective(120) having optics and an optic axis (124) and configured to focus theoptics at a focal plane (122) and to move along the optic axis (124); acoupler (132) configured to couple the illumination source (104) to theobjective (120) at a coupling angle, to move the illumination source(104) with the objective (120) in a scan direction along the optic axis(124), and to restrict the illumination plane (110) to the focal plane(122) as the objective (120) and the illumination source (104) movealong the optic axis (124); and a detector (126) configured to detect atleast one projected image from the illuminated focal plane (122) throughthe objective (120); wherein at least the illumination source (104) andthe objective (120) are tilted at a tilt angle.
 2. (canceled) 3.(canceled)
 4. The system of claim 1 wherein the coupling angle isapproximately orthogonal.
 5. The system of claim 1 wherein theillumination source (104) is configured to generate the thin light blade(106) at an illumination angle between the illumination plane (110) anda reference plane (134).
 6. The system of claim 1 wherein theillumination angle comprises an angle between approximately 30 and 60degrees, and the reference plane (134) comprises a sample referenceplane.
 7. The system of claim 1 further comprising an objectivepositioner (202) configured to move the objective (120) in and out alongthe optic axis (124).
 8. The system of claim 7 wherein the objectivepositioner (202) further is configured to move the objective (120),coupler (132), and illumination source (104) in the scan direction alongthe optic axis (124).
 9. The system of claim 1 further comprising arotational positioner (206) configured to rotate at least the objective(120), the coupler (132), and the illumination source (104) about arotational axis.
 10. The system of claim 9 wherein the rotational axiscomprises at least one member of a group consisting of the optic axis(124) and an illumination axis (112).
 11. The system of claim 1 furthercomprising a vertical positioner (210) configured to raise and lower atleast the objective (120), the coupler (132), and the illuminationsource (104).
 12. The system of claim 1 further comprising a processor(204) configured to control movement of at least rotational and verticalpositions of at least the objective (120), the coupler (132), and theillumination source (104).
 13. (canceled)
 14. A microscope assembly(102) for imaging samples (136) comprising: an illumination source (104)configured to generate a thin light blade (106) to an illumination plane(110); an objective (120) comprising a lens and an optic axis (124) andconfigured to focus the lens at a focal plane (122) along the optic axis(124); a coupler (132) configured to couple the illumination source(104) and the objective (120) and to restrict the illumination plane(110) to the focal plane (122) as the objective lens moves the focalplane (122) along the optic axis (124); and a detector (126) configuredto detect at least one projected image from the illuminated focal plane(122) through the objective (120).
 15. (canceled)
 16. (canceled)
 17. Themicroscope assembly (102) of claim 14 wherein the illumination source(104) comprises fiber configured to carry a beam of light and an opticassembly (116) configured to focus the beam of light into the thin lightblade (106) and to the illumination plane (110).
 18. (canceled)
 19. Themicroscope assembly (102) of claim 14 further comprising a positionerconfigured to move the microscope assembly (102) in a scan directionalong the optic axis (124).
 20. The microscope assembly (102) of claim14 further comprising a positioner configured to move the microscopeassembly (102) in a scan direction along a reference plane (134). 21.The microscope assembly (102) of claim 17 wherein at least theillumination source (104) and the objective (120) are tilted at a tiltangle.
 22. The microscope assembly (102) of claim 14 wherein the coupler(136) is configured to couple the illumination source (104) and theobjective (120) at a coupling angle that is approximately orthogonal.23. (canceled)
 24. (canceled)
 25. (canceled)
 26. The microscope assembly(102) of claim 14 further comprising a rotational positioner (206)configured to rotate the microscope assembly (102) about a rotationalaxis.
 27. The microscope assembly (102) of claim 26 wherein therotational axis comprises at least one member of a group consisting ofthe optic axis (124) and an illumination axis (112).
 28. (canceled) 29.The microscope assembly (102) of claim 14 wherein the processor (204)further is configured to control at least rotational and verticalpositions of the microscope assembly (102).
 30. (canceled) 31.(canceled)
 32. A method for imaging comprising: providing anillumination source (104) configured to generate a thin light blade(106) to an illumination plane (110) and comprising: providing fiberconfigured to carry a beam of light; and providing an optic assembly(114) configured to focus the beam of light into the thin light blade(106) and to the illumination plane (110); providing an objective (120)having optics and an optic axis (124), the objective configured to focusthe optics at a focal plane (122) and to move along the optic axis(124); coupling the illumination source (104) to the objective (120) ata coupling angle enabling moving the illumination source (104) with theobjective (120) in a scan direction along the optic axis (124) andrestricting the illumination plane to the focal plane as the objectiveand the illumination source (104) move along the optic axis (124);tilting at least the coupled illumination source (104) and the objectiveat a tilt angle in a synchronized movement; and detecting at least oneprojected image from the illuminated focal plane through the objective(120).
 33. (canceled)
 34. (canceled)